A fundamental but unsolved question in neuroscience is how specific connections between brain cells (neurons) underlie information processing in the neural circuits. Even the smallest local circuit in the cerebral cortex consists of tens of thousands of neurons, each making thousands of connections. Perhaps the biggest reason we don't understand the cerebral cortex is that we don't have an actual wiring diagram of any single cortical circuit. But even if we had a wiring diagram, we would need to know what each neuron in a circuit is doing: its physiology. In this proposal we plan to study neurons in the visual cortex whose responses to sensory stimuli have been characterized with new imaging techniques. This will allow us to see the function of literally every neuron in a cube 1/2 millimeter on a side. We will then use high-resolution and high-throughput anatomical techniques to uncover some of the connections between these functionally characterized neurons. Recent advances in functional imaging and serial-section electron microscopy allow us to study this difficult problem. We will address these questions in the mouse visual cortex, an emerging model of visual processing that is amenable to genetic manipulation and in vivo imaging techniques. We will use two- photon calcium imaging to see the activity of neurons in a functioning local circuit. We will then use large- scale serial-section electron microscopy to trace circuits in the same piece of cortex. By combining these methods, we will collect data sets that provide a physiological and structural overview of a well-studied brain circuit. It is now possible to study these circuits on their own terms: in all of their complexity and with data sets that are in many senses complete. PUBLIC HEALTH RELEVANCE: Many of the neurological and psychiatric diseases with the largest impact on public health-Alzheimer's disease, stroke, epilepsy, and autism-are functional disorders that likely have correlates in disordered brain connections. The proposed studies will characterize the functional connectivity of brain circuits with unprecedented resolution and completeness. In mouse models of functional brain disorders, the approaches we develop will greatly improve our ability to study the relationship between altered connections and functional deficits.